Human growth hormone (hGH) is a single chain polypeptide hormone containing 191 amino acids and two disulfide bonds. hGH is synthesized by the somatotropic cells of the anterior pituitary and plays an important role in somatic growth through its effects on the metabolism of proteins, carbohydrates, and lipids. At least four distinct biological activities have been ascribed to the mammalian growth hormone molecule, viz., (1) growth promotion, (2) lactogenic activity, (3) diabetogenic activity, and (4) insulin-like activity. This hormone has been used successfully in replacement therapies to treat pituitary dwarfism. Because of its anabolic properties, hGH is considered to be a candidate for use in the treatment of a variety of other medical conditions. However, its effects on carbohydrate metabolism are viewed as potential limitations in its utility in certain situations. As noted, hGH is reported to possess an early insulin-like activity which, in laboratory animals, causes a drop in serum glucose and a drop in serum free fatty acids. In addition, hGH has a diabetogenic activity which is observed several hours after administration of the hormone to test animals. In response to an oral glucose challenge, both serum glucose and insulin levels are elevated with a concomitant insulin resistant glucose intolerance. Although the data are limited in adult human subjects treated with hGH, the potential exists that the same metabolic perturbations may occur. For this reason, a non-diabetogenic form of hGH that retains its anabolic properties is highly desirable for use in treating adult humans.
We have discovered that the structure of growth hormones can be modified to eliminate or substantially reduce both the insulin-like and diabetogenic effects with retention of all or a portion of the growth hormone anabolic properties. It is to such a class of compounds that this invention is directed.
It has long been a research goal to separate these activities and to isolate a growth hormone "active core" having only or dominantly anabolic activity. Although there are a number of reports in the literature describing various modifications of human growth hormone (hGH), the goal of isolating the growth hormone "active core" has remained unfulfilled.
Three naturally-occurring, proteolytically cleaved, two-chain forms of human growth hormone have been characterized and are designated 24K, .alpha..sub.2, and .alpha..sub.3. These derivatives are described in a review article by Lewis et al., Rec. Progr. Horm. Res. 36, 477-508 (1980). The 24K form, which has been isolated from the pituitary, results from a single cleavage at Phe.sub.139. The .alpha..sub.2 form has been cleaved so as to remove residues 135 through 140, and the .alpha..sub.3 form is lacking residues 135 through 146. These derivatives in general show a potentiation of growth stimulating activity as well as an increase in lactogenic activity.
In addition to the naturally occurring derivatives, there is an extensive literature on the use of enzymes to proteolytically modify human growth hormone. Included among enzymes that have been used are trypsin, chymotrypsin, plasmin, thrombin, subtilisin, bromelain, fibrinolysin, and pepsin.
The use of trypsin to modify human growth hormone was first reported in 1965 by Li and Samuelsson, Mol. Pharmacol. 1, 47-52 (1965). These authors treated human growth hormone with trypsin for varying lengths of time and then tested the biological activities of the digests. They found that digestion for up to 30 minutes resulted in little loss of growth-promoting activity as measured by the rat tibia assay or little loss of lactogenic activity as measured by the pigeon crop sac assay. Longer digestion times resulted in a gradual loss of growth-promoting activity and an abrupt loss of lactogenic activity. More recently, Graf et al., J. Biol. Chem. 257, 2365-2369 (1982), have characterized a product of the trypsinolysis of human growth hormone as a derivative lacking residues 135 through 145. This derivative has similar properties to the intact hormone in two receptor-binding assays, a radioimmunoassay, and the rat tibia assay.
The enzyme plasmin has been extensively used to modify human growth hormone. Mills et al., Endocrinol. 102, 1366-1376 (1978), report on the digestion of reduced and S-carbamidomethylated human growth hormone with the isolation of several fractions and derivatives including, (1) the S-carbamidomethylated fragment consisting of residues 1-134, (2) fragment 20-41, (3) fragment 95-134, (4) the carbamidomethylated fragment 1-134 joined in a noncovalent complex with the carbamidomethylated fragment 141-191, (5) the deamidated noncovalent complex of the carbamidomethylated fragment 1-134 and the carbamidomethylated fragment 141-191, (6) a 1:1 deamidated noncovalent complex of carbamidomethylated fragments 1-134, 42-134, and 141-191, (7) Da, a fraction isolated from an anion exchange separation of the plasmin digest, (8) Db, a second, more acidic fraction isolated from the same anion exchange column, and (9) Dc, a third, more acidic fraction isolated from the same anion exchange column. Da and Db are reported to be equipotent with human growth hormone in the rat weight gain assay for growth-promoting activity. In a second paper from the same group, Reagan et al., Endocrinol. 102, 1377-1386 (1978), these derivatives are more extensively characterized biologically. Derivatives (4) and (5) were found to retain most of their growth-promoting activity in the rat weight gain assay and to be superpotent in a lactogenic assay. In contrast, derivative (6) had only about 1/3 the growth-promoting activity relative to intact human growth hormone but was equipotent in the lactogenic assay. Fraction (7) retained the growth-promoting activity and insulin-like activity as measured by glucose oxidation in rat epididymal adipose tissue, and exhibited slightly higher lactogenic activity. Fraction (8) retained the growth-promoting activity but lost about 1/2 of the insulin-like activity. Fraction (9) retained about 44% of the growth-promoting activity but was not tested for other biological activities. The unfractionated plasmin digest was found to have about 1/2 the growth-promoting activity and 1/2 the insulin-like activity of intact human growth hormone and about 3/4 of the lactogenic activity. The diabetogenic activity of some of these derivatives was tested in obese ob/ob [Reagan, Diabetes 27, 883-888 (1978)]. The unfractionated plasmin digest was found to retain 100% of the diabetogenic acitivity of intact human growth hormone, as was the S-carbamidomethylated derivative. Derivatives (4) and (6) likewise were found to be diabetogenic; however, derivative (2) had no diabetogenic activity.
Thrombin cleaves at a single site within the human growth hormone molecule, at Arg.sub.134, giving rise to a two-chain molecule with the two chains connected by a disulfide bond. Reduction and alkylation of the disulfide bond results in a noncovalent complex of the two chains. Several derivatives of this nature have been isolated from thrombin digests of human growth hormone [Mills et al., Endocrinol. 107, 391-399 (1980) and Reagan et al., Endocrinol. 109, 1663-1671 (1981)] including, (1) the derivative with the single clip at Arg.sub.134, (2) the S-carbamidomethylated noncovalent complex, (3) the S-aminoethylated noncovalent complex, (4) the S-carboxymethyl noncovalent complex, (5) the carbamidomethylated fragment 1-134, and (6) the carbamidomethylated fragment 135-191. Biological assays on these derivatives included the rat weight gain assay for growth-promotion, the oxidation of glucose as a measure of insulin-like activity, the obese ob/ob mouse assay for diabetogenic activity, and the N-acetyllactosamine synthase assay for lactogenic activity. Derivative (1) was fully potent in all assays except the diabetogenic assay in which it was not tested. Derivatives (2) and (3) were only 50% potent in the weight gain assay and 20% potent in the insulin-like assay, but were 75-80% potent in the lactogenic assay. Again, these two derivatives were not tested in the diabetogenic assay. Derivative (4) was found to have only 10% of the growth-promoting activity and 5% of the insulin-like activity but was fully potent in the lactogenic assay. Fragments (5) and (6) were found to have only slight activity in the weight gain assay and the insulin-like activity was very low. However, while fragment (5) possessed 25-50% of the diabetogenic activity of intact human growth hormone, fragment (6) had no detectible activity.
Li et al., J. Biol. Chem. 218, 41-52 (1956) studied the biological properties of the products of a chymotrypsin digest of human growth hormone. They found that increasing times of incubation resulted in a gradual loss of growth-promoting activity as measured by the rat tibia assay. However, even after 300 minutes of incubation, the digest still retained 75% of the activity of intact human growth hormone. An "active core" was obtained by dialysis of a chymotrypsin digest and was found to retain full potency.
Fibrinolysin has been used to modify human growth hormone by removal of a peptide consisting of residues 138 through 147 [Lewis et al., Biochem. Biophys. Res. Comm. 67, 617-624 (1975)]. This derivative was found to have greatly enhanced growth-promoting activity and lactogenic activity.
The digestion of human growth hormone with bromelain results in a mixture of three components, all three with a large fragment consisting of residues 1-134 and a smaller fragment consisting of either residues 143-191, 145-191, or 146-191. This mixture retained 70-80% of the growth-promoting activity of intact human growth hormone and 100% of the insulin-like activity [Mills et al., Biochim. Biophys. Acta 742, 169-174 (1983)]. If the above mixture is reduced and S-carbamidomethylated, all three of the alkylated smaller fragments can be isolated. These fragments could then be complemented with S-carbamidomethylated fragment 1-134 obtained from a thrombin digest resulting in a mixture of noncovalent complexes consisting of the alkylated analogs of the three derivatives. These were found to be sustantially less potent in both the growth-promoting and the insulin-like activity assays.
Pepsin was used to digest human growth hormone [Li, J. Gen. Physiol. 45, 169-178(1962)]. Growth-promoting activity was found to decrease with increasing periods of digestion, but even after 120 minutes, approximately 66% of the original activity remained. Lactogenic activity was fully retained at 60 minutes of digestion, but further digestion resulted in complete abolition of activity. An "active core" was isolated from the digestion by dialysis and retained 100% of the growth-promoting activity.
Limited hydrolysis of human growth hormone with subtilisin results in the formation of three two chain derivatives, S.sub.1, S.sub.2, and S.sub.3 [Lewis et al., Endocrinol. 101, 1587-1603 (1977)]. S.sub.1 consists of residues 1-139 connected through a disulfide bond to a fragment consisting of residues 150-191. S.sub.2 consists of residues 1-139 connected through a disulfide bond to a fragment consisting of residues 147-191. S.sub.3 is a deamidated derivative of S.sub.2. These three derivatives were tested in the rat tibia assay and were found to possess excellent growth-promoting activity. They were also tested for diabetogenic activity in dogs using oral glucose tolerance tests. S.sub.1 was found to be the most active in producing hyperglycemia and hyperinsulinemia. S.sub.2 and S.sub.3 were also diabetogenic but less so than S.sub.1.
There are two reports in the literature concerning a truncated analog of human growth hormone, prepared by recombinant DNA technology, which lacks the first 13 residues at the amino terminus. [Gertler et al., Endocrinol. 118, 720-726 (1986) and Ashkenazi et al., Endocrinol. 121, 414-419 (1987)]. This analog was found to inhibit the lactogenic activity of both human growth hormone and ovine prolactin in Nb2 cells and in explants from bovine lactating mammary gland. However, it did not have growth-promoting activity in the Nb2 cells line nor did it affect glucose uptake by the mammary gland explants. It did compete with radiolabeled human growth hormone for binding to Nb2 cells, IM-9 cells, the microsomal fraction from lactating bovine mammary gland, and the the microsomal fraction from rat liver, but with a much lower affinity.
Other growth hormones, primarily bovine, ovine, and rat have been modified proteolytically using many of the same enzymes as used for human growth hormone and their biological properties have been studied. The results are generally the same as those obtained with human growth hormone, i.e., proteolytic modification of the hormones resulted in an alteration of the biological properties. Only two examples will be given here, one using rat growth hormone and proteolysis by trypsin and the other using bovine and ovine growth hormone and proteolysis by trypsin. Maciag et al., J. Biol. Chem. 255, 6064-6070 (1980), found that limited trypsin hydrolysis of rat growth hormone resulted in the generation of two fragments, one consisting of residues 1-95 and residues 134-191 linked by a disulfide bond, and the other fragment consisting of residues 96-133. These two derivatives were examined for their abilities to interact with isolated hepatocytes and to stimulate growth in the rat tibia assay. The larger fragment displayed binding properties similiar to those of intact growth hormone but had little or no growth-promoting activity. The smaller fragment interacted weakly with growth hormone receptor sites but possessed significant growth-promoting activity.
Graf and Li, Biochem. 13, 5408-5415 (1974) digested both bovine and ovine growth hormones with trypsin and isolated a fragment from each digest that corresponded to residues 96-133 and a fragment from the bovine growth hormone digest that corresponded to residues 151-191. These derivatives were tested in the rat tibia assay for growth-promoting activity and all three were found to have measurable activity.
Thus, many derivatives of human growth hormone have been described resulting in alterations of the biological properties of the molecule. However, none of these derivatives involves both a truncation at the N-terminus as well as a cleavage or deletion in the large loop. Furthermore, none of these derivatives retains substantially all of the anabolic activity while having substantially diminished insulin-like and diabetogenic activities.